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  • Author: C. LUTWAK-MANN x
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The purpose of the preliminary experiments described below was to label mammalian spermatozoa with 32 P to such a degree that they could afterwards be used for a quantitative follow-up of the so-called leakage of intracellular sperm compounds, such as is known to accompany sperm 'ageing' or 'senescence' (Mann, 1964). The approach was twofold, by means of labelling in vivo and in vitro.

The in-vivo study, in which four buck rabbits were injected subcutaneously with inorganic [32 p]phosphate (1 mc/animal), included radio-activity assays by liquid scintillation counting of urine, blood plasma, blood corpuscles, seminal plasma and spermatozoa. In the urine and blood plasma, specific radio-activity, high at first, declined rapidly, reaching a very low level by the end of the first month. In the blood corpuscles, there was first a decline in radio-activity which, however,

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Comparisons of the antigenic and chemical composition of pig blood and follicular fluid are reported. Follicular fluid was found to contain less protein, protein-bound orcinol-reactive carbohydrate, reducing sugar and sialic acid, but a little more acid-soluble phosphate, than blood serum. Fructose and ergothioneine were not present in appreciable amounts in either follicular fluid or blood serum

Immunoelectrophoretic analysis showed that both follicular fluid and blood serum contained at least ten different antigenic components. Most of these were common to both but two of the serum antigens were not found in the follicular fluid. The follicular fluid antigen absent from serum appears to be fibrinogen.

Washings from the uteri of adult non-pregnant pigs contained at least six antigens most of which were not found in either follicular fluid or blood.

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The action of various agents was investigated on 6-day-old rabbit blastocysts in vivo and in vitro. The blastocysts were examined by the flat-mount technique. The substances studied included antimetabolites (2-deoxyglucose, 2-deoxyglucose-6-phosphate, 2-deoxygalactose, 6-mercaptopurine and 6-mercaptopurine riboside, ethionine, isoniazid, analogues of vitamin B12), enzyme inhibitors (dl-glyceraldehyde, salicylate, bromoacetylcarnitine, p-chloromercuribenzoate, fluoride), antimitotic agents (colcemid, aminopterin), cytostatic agents (actinomycin D, cytochalasin B), metabolites (DNA, glucose-6-phosphate, 2-deoxyribose, galactose), and hormones (polyoestriol phosphate, growth hormone). The influence of anoxia was also investigated, with special reference to temperature.

Agents which, under the experimental conditions laid down in this study, exerted clearly recognizable effects in vivo and in vitro, were 2-deoxyglucose, 6-mercaptopurine riboside, colcemid, isoniazid, aminopterin and DNA. Agents that were found to act in vitro only, were 2-deoxygalactose, glyceraldehyde, salicylate, bromoacetylcarnitine, 2-deoxyglucose-6-phosphate, actinomycin D, cytochalasin B, p-chloromercuribenzoate, and anoxia. An agent inert in vitro, but conducive (with longer exposure) to embryonic death in vivo, was polyoestriol phosphate. Growth hormone too, was inactive in vitro, but produced a slight effect in vivo. No effect was demonstrable in rabbit blastocysts following treatment with ethionine, analogues of vitamin B12, fluoride, glucose-6-phosphate, 2-deoxyribose, galactose.

Blastocysts, obtained from rabbits that had been subjected to embryotoxic agents (2-deoxyglucose, colcemid), were capable of recovery and further growth in vitro when the damage incurred in vivo was of a minor to moderate degree. Blastocysts moderately damaged by maternal 6-mercaptopurine or 6-mercaptopurine riboside treatment did not recover significantly; however, blastocysts more severely affected by these two compounds in vivo deteriorated further after incubation in vitro. Pretreatment of rabbits with polyoestriol phosphate yielded blastocysts which developed subsequently in vitro particularly well.

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(I) Solutions of labelled ions 32PO4 3-, 35SO4 2-, 24Na1+, 42K1+ and 131I,1- were administered parenterally to pregnant rabbits on Days o to 12 of gestation (the day of mating being designated o). Several experiments were also done with adult oestrous rabbits. Measurements of radioactivity were made at 45 min after ion injection. Results were expressed as the proportion, X IO-6, of the total radioactive ion injected, found present per mg tissue or fluid, fresh weight. (2) The following findings were made in normal, pregnant rabbits: (a) in the free-lying 6-day blastocyst values for all ions were low, that for PO4 3- being lowest, and, moreover, they showed a marked degree of divergence; this contrasted with the much higher and closely grouped values in the I2day foetus; (b) in the fluid withdrawn from the blastocoelic cavity on Days 7 to IO values for PO4 3-, SO4 2- and Na1+ rose to a peak on Day 8, and declined on Days 9 and IO; (c) in the endometrial secretion all ion values greatly exceeded those in the free-lying blastocysts and in the blastocyst fluid, and remained relatively stable during Days 6 to 12 of pregnancy; (d) placental tissue retained more than twice the amount of PO4 3- found in the inter-implantation segments of the endometrium, a phenomenon absent or much less prominent with the other ions; (e) all ions except K1+ showed higher values in the endometrium and secretion of non-pregnant rabbits than in the progestational phase of pregnancy; (f) exceptionally high PO4 3- values were typical of the endometrium and secretion on Days 2 and 3 of gestation. (3) The incorporation of 32PO4 3- was studied more extensively than that of the other ions, in pregnant rabbits treated with oestrogens (stilboestrol, oestradiol benzoate), certain purine analogues (6-mercaptopurine, 8-azaguanine) and trypan blue.

In pregnant rabbits which had received single doses of stilboestrol or oestradiol benzoate 36 hr before 32PO4 3- injection, there was a depression in PO4 3- uptake on Days 3 and 4 in the endometrium and secretion; this was in disparity with the enhanced ion uptake evident from Day 5 onwards. In placental tissue a rise above normal occurred only on Day 7, and was followed by a progressive decline from Day 8 onwards, being most marked in the foetal placenta at 12 days. Retention of PO4 3- was distinctly increased in the 6-day blastocysts; on the other hand, very low values were recorded in 12-day foetuses. In the blastocyst fluid the peak at 8 days was obliterated owing to higher values on Days 7, 9 and 10.

Suitably timed treatment of pregnant rabbits with mercaptopurine, azaguanine, or trypan blue, produced no changes in ion incorporation in the endometrium or secretion. Values for the 6-day blastocyst remained unaltered. A small but distinct depression in ion retention in the 12-day foetus and placenta followed treatment alike with the purine analogues and trypan blue. (4) In the cervix or vagina no changes were observed in ion uptake analogous to those recorded for endometrial mucosa and secretion. (5) The significance of this experimental approach to the problem of blastocyst-uterine relationships is discussed with respect to the functional organization in the pre-implantation blastocyst, the diurnal shifts in the uterine environment, the differential response to oestrogen treatment of uterine tissues depending upon their nature and the period of gestation, and the impaired ability of damaged foetus or placental tissue to retain ions entering from the maternal bloodstream.